At present, near the completion of the genome project, the focus of the research has rapidly shifted from gene structural analysis to gene functional analysis. It is believed that an intracellular protein does not function singly, but expresses its function cooperatively by interacting with various protein factors, nucleic acids, low-molecular species, and cell-membrane components, to biologically function as the sum of their interactions.
One of main subjects in the post-genome project is to analyze the relation between structure and function of various protein factor complexes. Results obtained from the analyses are expected to provide very important knowledge in wide areas covering basic biological studies, including structural biology and biochemistry, elucidation of the relation between the gene translation product and the etiology in the medical field, and the development of medicines.
As a method for carrying out, in vitro, the protein-synthesis reaction, a so-called “method for cell-free protein synthesis” or the like has been studied actively (Japan Patent Laid-Open Hei 6-98790, Japan Patent Laid-Open Hei 6-225783, Japan Patent Laid-Open Hei 7-194, Japan Patent Laid-Open Hei 9-291, Japan Patent Laid-Open Hei 7-147992). Methods can include components containing ribosomes or the like that can function as an intracellular original protein-translating device that are extracted from an organism, and a translation template, amino acids as substrate, energy sources, various ions, a buffer, and other effective factors that are added to the extract to synthesize a protein in vitro.
A cell extract or biological tissue extract for the protein synthesis for a reaction system for the cell-free protein system, i.e., a cell-free protein synthesis system, Escherichia coli, wheat embryo, rabbit reticulocyte, and so on are used. The cell-free protein synthesis system has properties comparable to a living cell with respect to “peptide synthesis rate” and “accuracy of translation reaction,” and is advantageous because it does not require any complex chemical reaction step or cell culture step. Therefore, a practical system has been developed for it. Generally, however, an extract from cells of an organism has only a very unstable ability to synthesize a protein, so that protein synthesis efficiency is low. In addition, the quality of the cell extract is significantly reduced during storage. Therefore, the amount of a product obtained from the cell-free protein synthesis system is small and can be detected only by radioisotope labelling or the like. Thus, the cell-free protein synthesis system can not be used as a practical method for producing a protein.
Conventional efficient cell-free protein synthesis methods includes the consecutive cell-free protein synthesis method developed by Spirin et al. [Spirin, A., et al., (1993) Methods in Enzymology, 217, 123-142]. They added an expression plasmid having an inserted objective gene to a transcription-translation coupled cell-free protein synthesis system using a cell extract prepared from Escherichia coli, wheat embryo, or rabbit reticulocyte. Spirin et al. showed that the protein synthesis system permits efficiently synthesizing a protein, and they reported that the consecutive cell-free protein synthesis method using a transcription-translation coupled cell-free protein synthesis system using the E. coli extract provides about 1 mg of product per 1 ml of the reaction mixture.
However, the cell-free protein synthesis system using Escherichia coli demonstrates a high protein synthesis ability when the plasmid is in a circular form and using an expression plasmid into which the objective gene was inserted as a template. When a linear plasmid or a linear transcription template constructed by the polymerase chain reaction (PCR) method is used, however, the template DNA is degraded by a DNase in the contaminated Escherichia coli extract in a time as short as 2 h or so using Escherichia coli in the cell-free protein synthesis system. Therefore, the amount of protein that can be synthesized is reduced to the level of the conventional batch method, i.e., several tens μg or so per 1 ml of the reaction system. Thus, a cell extract or tissue extract, for the cell-free protein synthesis, prepared from E. coli, wheat embryo, or rabbit reticulocyte, by an existing method, is contaminated with nucleases, translation inhibition protein factors, proteases, and so on, and these contaminants deactivate or inactivate the translation reaction system during the protein synthesis reaction [Ogasawara, T., et al., (1999) EMBO J., 18, 6522-6531]. Therefore, any protein synthesis system using the cell extract or tissue extract gives a low synthesis efficiency, and the amount of protein obtained is small.
Recently, the inventors provided methods for solving the problems of these cell-free protein synthesis systems, as described in 1) cell extract preparation for cell-free protein synthesis and cell-free protein synthesis method (WO00/68412) and 2) template molecules having generality and efficient functions, and methods for using the same (WO01/27260). These methods remarkably (i.e., successfully) enhanced the protein synthesis efficiency with a wheat embryo cell-free protein synthesis system. The wheat embryo cell-free protein synthesis system developed by the inventors does not contain any nuclease or translation reaction inhibitors [Madin, K. et al., (2000) Proc. Natl. Acad. Sci. USA, 97, 559-564] (WO00/68412), and permits carrying out the efficient protein synthesis using a linear DNA as a transcription template, which has been difficult so far. All applications, patents, and publications mentioned above and throughout are incorporated in their entirety by reference herein.
On the other hand, a transcription template for the above efficient wheat embryo cell-free protein synthesis system is prepared by the template DNA construction method using an existing PCR method. In this method, primers were used which contain:
{1} the total region of a promoter site of a vector that is a recognition and binding site for RNA polymerase,
{2} a structure contributing to the translation amplification and the stabilization of mRNA, and {3} a part of ORF (open reading frame) of the objective gene, with {3} being downstream of {1} and {2}. Using these primers, the construction of a transcription template for the cell-free protein synthesis from the objective gene has been tried. Therefore, in an existing method for constructing a transcription template by the PCR method, in addition to the objective full-length transcription template, short DNA fragments having a promoter sequence are accumulated. Short DNA fragments accumulate because of non-specific DNA amplification occurring during the reaction. It is difficult to remove these DNA fragments. Therefore, if RNA is synthesized using “a PCR product using the above primers” as a transcription template, almost all of molecules obtained are low-molecular-weight RNAs that are non-specific transcription products of a short DNA. These RNA molecules contain RNA fragments having 5′-translation initiation sequence, and these are recognized as mRNAs and translated. As a result, in addition to translation products of the objective gene, a large amount of low-molecular-weight translation products are formed, which results in the reduction of the yield and purity of the objective translation product.
Thus, an existing method for constructing a translation template using a transcription template constructed by designing and using a 5′-end side primer containing a base sequence complementary to the full-length promoter had the following big faults:
1) the transcription efficiency of the objective gene is very low, and
2) the method gives a translation template containing many noises (errors).
In addition, it is believed that even with the transcription template constructed, the efficient protein synthesis by the transcription-translation coupled method using the conventional batch wheat embryo cell-free protein synthesis system was impossible because the optimal magnesium concentration for the transcription reaction and for the translation reaction are quite different from each other. Moreover, the low efficiency of the cell-free protein synthesis system is affected by four ribonucleoside triphosphates (4NTPs) that are remnants of transcription substrates and pyrophosphate that is a by-product, which strongly inhibit the translation reaction.
With the progress of the genome project, many gene structures have been elucidated now, and several tens of thousands of cDNA clones have been isolated. As the first step for the analyses of functions and structures of genes in the basic and applied sciences in the 21st century, for the proteome analysis, and for the creation of medicines, it is necessary to simply and efficiently synthesize proteins that are gene products from the large amount of genes. The elemental technology to do so requires {1} designing an mRNA keeping a high translation template activity, {2} the transcription template-constructing technique for synthesizing an mRNA, and {3} a simple cell-free translation technique using the transcription template-constructing technique.